16th International HIV Drug Resistance Workshop 12-16 June 2007, Barbados

MAPPING OF VICRIVIROC RESISTANCE MUTATIONS IN HIV-1 GP120 GENERATED BY IN VITRO SELECTION OF A PRIMARY HIV-1 ISOLATE IN PM-1 CELLS

Antivir Ther. 2007; 12:S14 (abstract no. 12)

RA Ogert, C Buontempo, L Wojcik, L Ba, P Buontempo, RO Ralston, JM Strizki and JA Howe
Schering-Plough Research Institute, Kenilworth, NJ USA

BACKGROUND: Vicriviroc is one of a new class of CCR5 receptor antagonists in development for HIV-1 therapy. Currently, only limited data are available on the development of viral resistance to this class of drug. In this study, we evaluated viruses containing chimeric gp120 envelopes from resistant and susceptible isolates to better define the phenotypic and genotypic correlates of resistance to CCR5 antagonists.

METHODS: A vicriviroc-resistant virus was generated by serial passaging of a clade B isolate (QZ4589) in the presence of increasing concentrations of vicriviroc in PM-1 cells for >6 months. HIV gp120 sequences from resistant cultures were amplified by DNA PCR, and amino acid changes in resistant envelopes were identified by comparison to the parental passage control virus. Homologous recombination of gp120 fragments into a QZ4589 gp160 or ADA gp160 expression vector was performed in BJ5183 E. coli, or by fragment swap into a replication competent AD8 molecular clone. Susceptibility studies were performed with a single-cycle and multi-cycle infection assays. Site-directed mutagenesis was performed to assess the contribution of individual and combined gp120 mutations on CCR5 antagonist susceptibility.

RESULTS: We identified 10 amino acid changes within gp120 associated with viral resistance. Full-length HIV-1 QZ4589 chimeric clones pseudotyped poorly; therefore, we recombined the C2-C5 gp120 fragment from a resistant env clone into an ADA gp160 expression vector; resulting in significantly enhanced luciferase signals. The QZ4589 C2-C5 ADA recombinant env was resistant to vicriviroc as evidenced by a decrease in the percent maximal inhibition observed. Chimeric envelopes containing only the V3 region of the QZ4589-resistant virus were completely susceptible to vicriviroc. Site-directed mutagenesis of individual amino acids revealed that three changes: R305K (V3), P363S (C3), and T467I (V5) restored partial susceptibility to vicriviroc. Envelopes containing the Q315R, A373T, N413T, and S437P mutations retained resistance to vicriviroc.

CONCLUSIONS: HIV-1 resistance to vicriviroc in the QZ4589 isolate maps to multiple amino-acid changes in the C2-V5 region. Single mutations within the V3, C3, and V5 regions restored partial, but not complete susceptibility to vicriviroc. Introduction of the QZ4589 V3-loop region into a heterologous background did not confer resistance to the chimeric envelope. This finding demonstrates that changes in the V3-loop are context dependent and alone are insufficient to cause vicriviroc resistance.

2007-06-12
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